Therapeutic agent and therapeutic method for periodontal diseases and pulpal diseases

ABSTRACT

The objects of the present invention are: to provide a therapeutic agent and a therapeutic method for periodontal diseases and pulpal diseases, a transplant for periodontal tissue regeneration, and a method for regenerating the periodontal tissue.
         According to the present invention, there are provided therapeutic agents for periodontal diseases and pulpal diseases which comprise neurotrophic factors as an active ingredient.

TECHNICAL FIELD

This invention relates to a therapeutic agent and a therapeutic methodfor periodontal diseases and pulpal diseases, a transplant forperiodontal tissue regeneration, and a method for regenerating theperiodontal tissue.

BACKGROUND ART

The periodontal tissue which is composed of gingiva, alveolar bone,periodontal ligament (periodontal membrane), cementum, dental pulp, etc.is essential for erecting teeth and maintaining their functions such asmastication and occlusion, and its damage or destruction will lead tothe loss of teeth. Consider, for example, a periodontal disease which isreportedly afflicting about 30 million people in Japan; as the diseaseprogresses, the periodontal, tissue becomes increasingly damaged ordestroyed resulting in tooth loss. To treat damaged or destroyedperiodontal tissue including the dental pulp, various methods comprisingmedication and surgical operation are being attempted but none of themedicaments and therapeutic methods are sufficiently effective toregenerate the damaged or destroyed periodontal tissue including thedental pulp.

Neurotrophic factors include brain-derived neurotrophic factor (BDNF),nerve growth factor (NGF), neurotrophin-3 (NT-3) and neurotrophin-4/5(NT-4/5) and are involved in differentiation, survival, regeneration andfunctional maintenance of neuron. BDNF and NT-4/5 bind to thehigh-affinity receptor TrkB (tropomyosin receptor kinase B), NGF toTrkA, and NT-3 to TrkC.

BDNF, NGF and NT-3 are neurotrophic factors mostly present in the brainand the efficacy of BDNF and NGF has been demonstrated in experimentswith animals of various disease models such as a motor neuropathy model,a Parkinson's disease model, and an Alzheimer disease model. Inparticular, BDNF is expected as an effective therapeutic drug for motorand peripheral nervous diseases such as amyotrophic lateral sclerosis(ALS) and peripheral neuropathies due to diabetes and chemotherapeuticagents, and for diseases involving the central nervous system such asthe Alzheimer disease, the Parkinson's disease, and retina-relateddiseases.

These neurotrophic factors are said to play an important role not onlyin the central nervous system but also in the peripheral nervous system.It has been reported that the expression of BDNF, NGF, NT-3, TrkC andTrkA increased during the healing process of fractured mouse ribs (K.Asaumi et al., Bone, Vol. 26, No. 6, 625-633, 2000) and that BDNF, NGFand NT-3 enhanced the proliferation of mouse periodontal ligament cells(Y. Tsuboi et al., J Dent Res 80(3):881-886, 2001). However, there areno detailed reports on the behavior of those neurotrophic factors in theperiodontal tissue and pulp tissue.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The objects of the present invention are: to provide therapeutic agentand a therapeutic method for periodontal diseases and pulpal diseases, atransplant for periodontal tissue regeneration, and a method forregenerating the periodontal tissue.

Means for Solving the Problems

The present inventors made intensive studies with a view to solving theabove-mentioned problems and found that neurotrophic factors enhancedthe proliferation of human periodontal ligament cells, the expression ofmRNA for bone-related proteins, and the regeneration of the periodontaltissue in dog models of a lesion in furcation regions. The presentinvention has been accomplished on the basis of these findings.

Thus, according to the present invention, there is provided atherapeutic agent for periodontal diseases which comprises aneurotrophic factor as an active ingredient.

The therapeutic agent of the present invention preferably regeneratesthe periodontal tissue.

The therapeutic agent of the present invention preferably regeneratesthe cementum, periodontal ligament, alveolar bone, or dental pulp.

The therapeutic agent of the present invention preferably prevents theapical invasion of gingival epithelium along the dental root surface.

The therapeutic agent of the present invention preferably enhances theproduction of repaired dentin in the pulp cavity. It is also preferredfor the therapeutic agent to enhance the addition of repaired dentin tothe inner surfaces of the pulp cavity.

In the therapeutic agent of the present invention, the neurotrophicfactor is preferably a brain-derived neurotrophic factor, a nerve growthfactor, neurotrophin-3, or neurotrophin-4/5.

According to another aspect of the present invention, there is provideda transplant for periodontal tissue regeneration which comprises aneurotrophic factor.

The transplant of the present invention preferably regenerates theperiodontal tissue.

The transplant of the present invention preferably regenerates thecementum, periodontal ligament, alveolar bone, or dental pulp.

The transplant of the present invention preferably prevents the apicalinvasion of gingival epithelium along the dental root surface.

The transplant of the present invention preferably enhances theproduction of repaired dentin in the pulp cavity. It is also preferredfor the transplant to promote the addition of repaired dentin to theinner surfaces of the pulp cavity.

In the transplant of the present invention, the neurotrophic factor ispreferably a brain-derived neurotrophic factor, a nerve growth factor,neurotrophin-3, or neurotrophin-4/5.

According to still another aspect of the present invention, there isprovided a method for regenerating the periodontal tissue whichcomprises using a neurotrophic factor.

The regenerating method of the present invention preferably regeneratesthe periodontal tissue.

The regenerating method of the present invention preferably regeneratesthe cementum, periodontal ligament, alveolar bone, or dental pulp.

The regenerating method of the present invention preferably prevents theapical invasion of gingival epithelium along the dental root surface.

In the regenerating method of the present invention, the neurotrophicfactor is preferably a brain-derived neurotrophic factor, a nerve growthfactor, neurotrophin-3, or neurotrophin-4/5.

According to yet another aspect of the present invention, there isprovided a therapeutic method for periodontal disease which comprisesadministering a therapeutically effective amount of a neurotrophicfactor to a subject who is suffering or prone to suffer from thedisease.

The therapeutic method of the present invention preferably regeneratesthe periodontal tissue.

The therapeutic method of the present invention preferably regeneratesthe cementum, periodontal ligament, alveolar bone, or dental pulp.

The therapeutic method of the present invention preferably prevents theapical invasion of gingival epithelium along the dental root surface.

The therapeutic method of the present invention preferably enhances theproduction of repaired dentin in the pulp cavity. It is also preferredfor the method to enhance the addition of repaired dentin to the innersurfaces of the pulp cavity.

In the therapeutic method of the present invention, the neurotrophicfactor is preferably a brain-derived neurotrophic factor, a nerve growthfactor, neurotrophin-3, or neurotrophin-4/5.

According to a further aspect of the present invention, there isprovided use of a neurotrophic factor for producing a medicament to beused in the therapy of periodontal diseases.

The medicament preferably regenerates the periodontal tissue, inparticular, the cementum, periodontal ligament, alveolar bone, or dentalpulp. The medicament preferably prevents the apical invasion of gingivalepithelium along the dental root surface. The medicament preferablyenhances the production of repaired dentin in the pulp cavity. It isalso preferred for the medicament to enhance the addition of repaireddentin to the inner surfaces of the pulp cavity. The neurotrophic factoris preferably a brain-derived neurotrophic factor, a nerve growthfactor, neurotrophin-3, or neurotrophin-4/5.

According to yet a further aspect of the present invention, there isprovided a repaired dentin morphogenesis enhancer comprising aneurotrophic factor as an active ingredient. The neurotrophic factor ispreferably a brain-derived neurotrophic factor, a nerve growth factor,neurotrophin-3, or neurotrophin-4/5. Repaired dentin is preferably addedto the inner surfaces of the pulp cavity.

According to another aspect of the present invention, there is provideda therapeutic method for pulpal disease which comprises administering atherapeutically effective amount of a neurotrophic factor to a subjectwho is suffering or prone to suffer from the disease in order to enhancethe morphogenesis of repaired dentin. The neurotrophic factor ispreferably a brain-derived neurotrophic factor, a nerve growth factor,neurotrophin-3, or neurotrophin-4/5. Repaired dentin is preferably addedto the inner surfaces of the pulp cavity.

According to yet another aspect of the present invention, there isprovided use of a neurotrophic factor for producing a medicament to beused for enhancing the morphogenesis of repaired dentin. Theneurotrophic factor is preferably a brain-derived neurotrophic factor, anerve growth factor, neurotrophin-3, or neurotrophin-4/5. Repaireddentin is preferably added to the inner surfaces of the pulp cavity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of electrophoretograms showing the expression of mRNAfor BDNF and TrkB in HPL cells and human periodontal ligament; the laneat the left end of each electrophoretogram is the marker; (A) shows theexpression of mRNA (613 bp) of glyceraldehyde 3-phosphate dehydrogenase(GAPDH) in human periodontal ligament and HPL cells; (B) shows theexpressions of mRNA (438 bp) of BDNF and mRNA (434 bp) of TrkB in humanperiodontal ligament; (C) shows the expression of mRNA for BDNF and TrkBin HPL cells.

FIG. 2A shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of BDNF and the amount of ALPase mRNA (381 bp)expression in HPL cells; HPL cells were all treated with BDNF at a finalconcentration of 50 ng/ml; the lane at the left end of theelectrophoretogram is the marker; the vertical axis of the graph plotsthe relative amount of mRNA expression for each exposure time, with theamount of mRNA expression for exposure time zero being taken as unity;the horizontal axis of the graph plots the exposure time of BDNF; thevertical lines on the bars in the graph each represents the range ofmean±standard deviation; ** means the statistically significantdifference at p<0.01 (in t-test).

FIG. 2B shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of BDNF and the amount of OPN mRNA (532 bp)expression in HPL cells; HPL cells were all treated with BDNF at a finalconcentration of 50 ng/ml; the lane at the left end of theelectrophoretogram is the marker; the vertical axis of the graph plotsthe relative amount of mRNA expression for each exposure time, with theamount of mRNA expression for exposure time zero being taken as unity;the horizontal axis of the graph plots the exposure time of BDNF; thevertical lines on the bars in the graph each represents the range ofmean±standard deviation; ** means the statistically significantdifference at p<0.01 (in t-test).

FIG. 2C shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of BDNF and the amount of BMP-2 mRNA (440 bp)expression in HPL cells; HPL cells were all treated with BDNF at a finalconcentration of 50 ng/ml; the lane at the left end of theelectrophoretogram is the marker; the vertical axis of the graph plotsthe relative amount of mRNA expression for each exposure time, with theamount of mRNA expression for exposure time zero being taken as unity;the horizontal axis of the graph plots the exposure time of BDNF; thevertical lines on the bars in the graph each represents the range ofmean±standard deviation; ** means the statistically significantdifference at p<0.01 (in t-test).

FIG. 2D is an electrophoretogram showing the relationship between theexposure time of BDNF and the amount of GAPDH mRNA expression in HPLcells; HPL cells were all treated with BDNF at a final concentration of50 ng/ml; the lane at the left end of the electrophoretogram is themarker.

FIG. 3A shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of BDNF and the amount of BMP-4 mRNA (339 bp)expression in HPL cells; HPL cells were all treated with BDNF at a finalconcentration of 50 ng/ml; the lane at the left end of theelectrophoretogram is the marker; the vertical axis of the graph plotsthe percentage of the amount of mRNA expression for each exposure time,with the amount of mRNA expression for exposure time zero being taken as100; the horizontal axis of the graph plots the exposure time of BDNF;the vertical lines on the bars in the graph each represents the range ofmean±standard deviation.

FIG. 3B shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of BDNF and the amount of OPG mRNA (736 bp)expression in HPL cells; HPL cells were all treated with BDNF at a finalconcentration of 50 ng/ml; the lane at the left end of theelectrophoretogram is the marker; the vertical axis of the graph plotsthe percentage of the amount of mRNA expression for each exposure time,with the amount of mRNA expression for exposure time zero being taken as100; the horizontal axis of the graph plots the exposure time of BDNF;the vertical lines on the bars in the graph each represents the range ofmean±standard deviation.

FIG. 4A shows by an electrophoretogram and a bar graph the relationshipbetween the dose at which BDNF was administered to HPL cells and theamount in which ALPase mRNA was expressed; the respective concentrationsof BDNF were allowed to act on the HPL cells for 24 hours; the lane atthe left end of the electrophoretogram is the marker; the vertical axisof the graph plots the relative amount of mRNA expression at each doseof BDNF, with the amount of mRNA expression for zero dose being taken asunity; the horizontal axis of the graph plots the concentration of BDNF(ng/ml); the vertical lines on the bars in the graph each represents therange of mean±standard deviation; ** means the statistically significantdifference at p<0.01 (in t-test).

FIG. 4B shows by an electrophoretogram and a bar graph the relationshipbetween the dose at which BDNF was administered to HPL cells and theamount in which OPN mRNA was expressed; the respective concentrations ofBDNF were allowed to act on the HPL cells for 12 hours; the lane at theleft end of the electrophoretogram is the marker; the vertical axis ofthe graph plots the relative amount of mRNA expression at each dose ofBDNF, with the amount of mRNA expression for zero dose being taken asunity; the horizontal axis of the graph plots the concentration of BDNF(ng/ml); the vertical lines on the bars in the graph each represents therange of mean±standard deviation; ** means the statistically significantdifference at p<0.01 (in t-test).

FIG. 4C shows by an electrophoretogram and a bar graph the relationshipbetween the dose at which BDNF was administered to HPL cells and theamount in which BMP-2 mRNA was expressed; the respective concentrationsof BDNF were allowed to act on the HPL cells for 24 hours; the lane atthe left end of the electrophoretogram is the marker; the vertical axisof the graph plots the relative amount of mRNA expression at each doseof BDNF, with the amount of mRNA expression for zero dose being taken asunity; the horizontal axis of the graph plots the concentration of BDNF(ng/ml); the vertical lines on the bars in the graph each represents therange of mean±standard deviation; * and ** mean the statisticallysignificant differences at p<0.05 and p<0.01, respectively (in t-test).

FIG. 4D is an electrophoretogram showing the relationship between thedose at which BDNF was administered to HPL cells and the amount at whichGAPDH mRNA (613 bp) was expressed.

FIG. 5 (A) is a bar graph showing the relationship between the dose atwhich BDNF was administered to HPL cells and the amount in which OPN wassecreted; the respective concentrations of BDNF were allowed to act onthe HPL cells for 12 hours; the vertical axis plots the amount of OPNsecretion (ng/ml) and the horizontal axis plots the concentration ofBDNF (ng/ml);

FIG. 5 (B) is a bar graph showing the relationship between the dose atwhich BDNF was administered to HPL cells and the amount in which BMP-2was secreted; the respective concentrations of BDNF were allowed to acton the HPL cells for 24 hours; the vertical axis plots the amount ofBMP-2 secretion (pg/ml) and the horizontal axis plots the concentrationof BDNF (ng/ml);

FIG. 5 (C) is a bar graph showing the relationship between the exposuretime of BDNF and the amount in which BMP-2 was secreted in HPL cells;the cells were treated with BDNF at a final concentration of 50 ng/ml;the vertical axis plots the amount of BMP-2 secretion (pg/ml) and thehorizontal axis plots the exposure time of BDNF; the vertical lines onthe bars in the graphs (A)-(C) each represents the range ofmean±standard deviation; ** means the statistically significantdifference at p<0.01 (in t-test).

FIG. 6 shows by means of a bar graph the relationship between the doseat which BDNF was administered and the ability of HPL cells and HGK tosynthesize DNA; the respective concentrations of BDNF were allowed toact on HPL cells and HGK for 24 hours; the vertical axis of each graphplots the relative ability to synthesize DNA at each dose of BDNF orbFGF, with the ability to synthesize DNA in the absence of BDNF or bFGF(i.e. at zero concentration of BDNF or bFGF) being taken as 100; thehorizontal axis plots the concentration of BDNF or bFGF (ng/ml); thevertical lines on the bars in the graphs each represents the range ofmean±standard deviation; * and ** mean the statistically significantdifferences at p<0.05 and p<0.01, respectively (in t-test); (A) showsthe ability to synthesize DNA in HPL cells, and (B) shows the ability tosynthesize DNA in HGK.

FIG. 7 (A) is a bar graph showing the relationship between the dose atwhich BDNF was administered to HPL cells and the amount in which type Icollagen was synthesized; the respective concentrations of BDNF wereallowed to act on HPL cells for 24 hours; the vertical axis plots theamount (μg/ml) in which type I collagen was synthesized and thehorizontal axis plots the concentration of BDNF (ng/ml);

FIG. 7 (B) is a bar graph showing the relationship between the exposuretime of BDNF and the amount in which type I collagen was synthesized;the cells were treated with BDNF at a final concentration of 50 ng/ml;the vertical axis plots the amount (μg/ml) in which type I collagen wassynthesized and the horizontal axis plots the exposure time of BDNF; thevertical lines on the bars in the graphs (A) and (B) each represents therange of mean±standard deviation; * and ** mean the statisticallysignificant differences at p<0.05 and p<0.01, respectively (in t-test).

FIG. 8 shows by means of a bar graph the relationship between the doseat which BDNF was administered to a dog model of class III furcationdefect and the regeneration of the cementum and the alveolar bone; thevertical axis plots the percent regeneration of the cementum or bone,and the horizontal axis plots the concentration of BDNF (μg/ml); thevertical lines on the bars in the graphs each represents the range ofmean±standard deviation; * and ** mean the statistically significantdifferences at p<0.05 and p<0.01, respectively (in t-test); (A) showsthe relationship with the percent regeneration of the cementum and (B)shows the relationship with the percent regeneration of the bone.

FIG. 9A is an optical microscopic view (×20) of a hematoxylin-eosinstained specimen of a bone defect at furcation and packed with aBDNF-free TERUPLUG® (CONTROL), prepared in Example 2.

FIG. 9B is a microscopic view (×20) of a bone defect at furcation andpacked with a transplant containing BDNF (5 μg/ml), prepared in Example2.

FIG. 10 is a partial enlarged view (×200) of the area right under thefurcation shown in FIG. 9B; in that area and in almost all parts of theexposed dental root surface, cementum had been regenerated with collagenfibers embedded therein and there was no invasion of epithelium.

FIG. 11A shows by a radioactivity band and a bar graph the amount atwhich NGF mRNA was expressed in HPL cells; the vertical axis of thegraph plots the amount of NGF mRNA expression relative to the amount ofGAPDH mRNA expression; in the graph, HGF designates gingivalfibroblasts, HPC, pulp cells, HSF, human skin fibroblasts, and HNB,human neuroblastoma cells.

FIG. 11B shows by a radioactivity band and a bar graph the amount atwhich TrkA mRNA was expressed in HPL cells; the vertical axis of thegraph plots the amount of TrkA mRNA expression relative to the amount ofGAPDH mRNA expression; in the graph, HGF designates gingivalfibroblasts, HPC, pulp cells, HSF, human skin fibroblasts, and HNB,human neuroblastoma cells.

FIG. 12 shows by means of a bar graph the effects of NGF on the amountof OPN mRNA expression in HPL cells; (A) is a graph showing the resultsof measuring the time course effect of NGF; the vertical axis of thegraph plots the relative amount of OPN mRNA expression for each exposuretime of NGF, with the amount of mRNA expression for exposure time zerobeing taken as unity; the horizontal axis of the graph plots theexposure time of NGF; all cells were treated with NGF at a finalconcentration of 100 ng/ml; (B) is a graph showing the results ofmeasuring the dose effect; the vertical axis of the graph plots therelative amount of OPN mRNA expression at each concentration of NGF,with the amount of mRNA expression at zero concentration being taken asunity; the horizontal axis of the graph plots the concentration of NGF(ng/ml); NGF exposure time was 24 hours in all cases.

FIG. 13 shows by means of a bar graph the effects of NGF on the amountof ALPase mRNA expression in HPL cells; (A) is a graph showing theresults of measuring the time course effect of NGF; the vertical axis ofthe graph plots the relative amount of ALPase mRNA expression for eachexposure time of NGF, with the amount of ALPase mRNA expression forexposure time zero being taken as unity; the horizontal axis of thegraph plots the exposure time of NGF; (B) is a graph showing the resultsof measuring the dose effect; the vertical axis of the graph plots therelative amount of ALPase mRNA expression at each concentration of NGF,with the amount of ALPase mRNA expression at zero concentration beingtaken as unity; the horizontal axis of the graph plots the concentrationof NGF (ng/ml).

FIG. 14 shows by means of a bar graph the effects of NGF on the amountof BMP-2 mRNA expression in HPL cells; (A) is a graph showing theresults of measuring the time course effect of NGF; the vertical axis ofthe graph plots the relative amount of BMP-2 mRNA expression for eachexposure time of NGF, with the amount of BMP-2 mRNA expression forexposure time zero being taken as unity; the horizontal axis of thegraph plots the exposure time of NGF; (B) is a graph showing the resultsof measuring the dose effect; the vertical axis of the graph plots therelative amount of BMP-2 mRNA expression at each concentration of NGF,with the amount of BMP-2 mRNA expression at zero concentration beingtaken as unity; the horizontal axis of the graph plots the concentrationof NGF (ng/ml).

FIG. 15 shows by means of a bar graph the relationship between the doseat which NGF was administered and the ability of HPL cells and HGK tosynthesize DNA; the respective concentrations of NGF were administeredto HPL cells and HGK for 24 hours; the vertical axis of each graph plotsthe relative ability to synthesize DNA at each dose of NGF, with theability to synthesize DNA at zero concentration of NGF being taken as100; the horizontal axis plots the concentration of NGF (ng/ml); (A)shows the ability to synthesize DNA in HPL cells, and (B) shows theability to synthesize DNA in HGK.

FIG. 16A shows by a radioactivity band and a bar graph the amount ofNT-3 mRNA expression in HPL cells; the vertical axis of the graph plotsthe relative amount of NT-3 mRNA expression, with the amount of GAPDHmRNA expression being taken as unity; in the graph, HGF designatesgingival fibroblasts, HPC, pulp cells, HSF, human skin fibroblasts, andHNB, human neuroblastoma cells.

FIG. 16B shows by a radioactivity band and a bar graph the amount ofTrkC mRNA expression in HPL cells; the vertical axis of the graph plotsthe relative amount of TrkC mRNA expression, with the amount of GAPDHmRNA expression being taken as unity; in the graph, HGF designatesgingival fibroblasts, HPC, pulp cells, HSF, human skin fibroblasts, andHNB, human neuroblastoma cells.

FIG. 17 is a bar graph showing the relationship between the dose atwhich NT-3 was administered and the ALPase activity in HPL cells; thevertical axis of the graph plots the ALPase activity (nmol/well) and thehorizontal axis plots the concentration of NT-3 (ng/ml).

FIG. 18 is a bar graph showing the relationship between the dose atwhich NT-3 was administered and the ability to synthesize DNA in HPLcells; the vertical axis of the graph compares by absorbance the abilityof HPL cells to synthesize DNA at various concentrations of NT-3; andthe horizontal axis plots the concentration of NT-3 (ng/ml).

FIG. 19A shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of NT-4/5 and the amounts in which mRNAs ofOPN and OCN were expressed in HPL cells; the final concentration ofNT-4/5 was adjusted at 50 ng/ml; the lane at the left end of eachelectrophoretogram is the marker; the vertical axis of each graph plotsthe relative amount of mRNA expression for exposure time, with theamount of mRNA expression for exposure time zero being taken as 100; thehorizontal axis of each graph plots the exposure time of NT-4/5; thevertical lines on the bars in each graph represent the range ofmean±standard deviation; in each graph, * and ** mean p<0.05 and p<0.01,respectively (statistical testing by t-test).

FIG. 19B shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of NT-4/5 and the amounts in which mRNAs ofBMP-2 and BMP-7 were expressed in HPL cells; the final concentration ofNT-4/5 was adjusted at 50 ng/ml; the lane at the left end of eachelectrophoretogram is the marker; the vertical axis of each graph plotsthe relative amount of mRNA expression for each exposure time, with theamount of mRNA expression for exposure time zero being taken as 100; thehorizontal axis of each graph plots the exposure time of NT-4/5; thevertical lines on the bars in each graph represent the range ofmean±standard deviation; in each graph, * and ** mean p<0.05 and p<0.01,respectively (statistical testing by t-test).

FIG. 19C shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of NT-4/5 and the amount in which mRNA ofALPase was expressed in HPL cells; it also shows by anelectrophoretogram the relationship between the exposure time of NT-4/5and the amount of GAPDH expression; the final concentration of NT-4/5was adjusted at 50 ng/ml; the lane at the left end of eachelectrophoretogram is the marker; the vertical axis of the graph plotsthe relative amount of mRNA expression for each exposure time, with theamount of mRNA expression for exposure time zero being taken as 100; thehorizontal axis of the graph plots the exposure time of NT-4/5; thevertical lines on the bars in the graph represent the range ofmean±standard deviation; * means p<0.05 (statistical testing by t-test).

FIG. 20 is a set of graphs showing the results of measuring the doseeffect of NGF on the expression of mRNA for various bone-relatedproteins (ALPase, BMP-2, DSPP, OPN, and OCN) in HP cells; the exposuretime of NGP was 24 hours; the vertical axis of each graph plots therelative amount of mRNA expression at each concentration of NGF, withthe amount of mRNA expression at zero concentration being taken asunity; the horizontal axis plots the concentration of NGF (ng/ml); thevertical lines on the bars in each graph represent the range ofmean±standard deviation.

FIG. 21 is a set of graphs showing the results of measuring the doseeffect of BDNF on the expression of mRNA for various bone-relatedproteins (ALPase, BMP-2, DSPP, type I collagen, OPN, and OCN) in HPcells; the vertical axis of each graph plots the relative amount of mRNAexpression at each concentration of BDNF, with the amount of mRNAexpression at zero concentration being taken as unity; the horizontalaxis plots the concentration of BDNF (ng/ml); the vertical lines on thebars in each graph represent the range of mean±standard deviation.

FIG. 22 is a set of graphs showing the results of measuring the doseeffect of NT-3 on the expression of mRNA for various bone-relatedproteins (ALPase, BMP-2, DSPP, OPN, and OCN) in HP cells; the verticalaxis of each graph plots the relative amount of mRNA expression at eachconcentration of NT-3, with the amount of mRNA expression at zeroconcentration being taken as unity; the horizontal axis plots theconcentration of NT-3 (ng/ml); the vertical lines on the bars in eachgraph represent the range of mean±standard deviation.

FIG. 23 is a set of graphs showing the results of measuring the doseeffect of NT-4/5 on the expression of mRNA for various bone-relatedproteins (ALPase, BMP-2, DSPP, type I collagen, OPN, and OCN) in HPcells; the vertical axis of each graph plots the relative amount of mRNAexpression at each concentration of NT-4/5, with the amount of mRNAexpression at zero concentration being taken as unity; the horizontalaxis plots the concentration of NT-4/5 (ng/ml); the vertical lines onthe bars in each graph represent the range of mean±standard deviation.

FIG. 24 is a set of bar graphs showing the relationship between the doseat which various neurotrophic factors (NGF, BDNF, NT-3, and NT-4/5) wereadministered and the ability of HP cells to synthesize DNA; therespective concentrations of the neurotrophic factors were allowed toact on HP cells for 24 hours; the vertical axis of each graph plots therelative absorbance at each dose of a neurotrophic factor, with theabsorbance without a neurotrophic factor (i.e. at zero concentration ofa neurotrophic factor) being taken as 100; the horizontal axis plots theconcentration of each neurotrophic factor (ng/ml); the vertical lines onthe bars in each graph represent the range of mean±standard deviation.

FIG. 25A shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of NGF and the amount of ALPase mRNAexpression in HMS cells; it also shows an electrophoretogram depictingthe relationship with the amount of GAPDH mRNA expression as control;HMS cells were all treated with NGF at a final concentration of 100ng/ml; the lane at the left end of each electrophoretogram is themarker; the vertical axis of the graph plots the percentage of theamount of mRNA expression for each exposure time of NGF, with the amountof mRNA expression for exposure time zero being taken as 100%; thehorizontal axis plots the exposure time of NGF.

FIG. 25B shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of NGF and the amount of OCN mRNA expressionin HMS cells; it also shows an electrophoretogram depicting therelationship with the amount of GAPDH mRNA expression; HMS cells wereall treated with NGF at a final concentration of 100 ng/ml; the lane atthe left end of each electrophoretogram is the marker; the vertical axisof the graph plots the percentage of the amount of mRNA expression foreach exposure time of NGF, with the amount of mRNA expression forexposure time zero being taken as 100%; the horizontal axis plots theexposure time of NGF.

FIG. 25C shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of NGF and the amount of OPN mRNA expressionin HMS cells; it also shows an electrophoretogram depicting therelationship with the amount of GAPDH mRNA expression; HMS cells wereall treated with NGF at a final concentration of 100 ng/ml; the lane atthe left end of each electrophoretogram is the marker; the vertical axisof the graph plots the percentage of the amount of mRNA expression foreach exposure time of NGF, with the amount of mRNA expression forexposure time zero being taken as 100%; the horizontal axis plots theexposuretime of NGF.

FIG. 25D shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of NGF and the amount of BSP mRNA expressionin HMS cells; it also shows an electrophoretogram depicting therelationship with the amount of GAPDH mRNA expression; HMS cells wereall treated with NGF at a final concentration of 100 ng/ml; the lane atthe left end of each electrophoretogram is the marker; the vertical axisof the graph plots the percentage of the amount of mRNA expression foreach exposure time of NGF, with the amount of mRNA expression forexposure time zero being taken as 100%; the horizontal axis plots theexposure time of NGF.

FIG. 25E shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of NGF and the amount of type I collagen mRNAexpression in HMS cells; it also shows an electrophoretogram depictingthe relationship with the amount of GAPDH mRNA expression; HMS cellswere all treated with NGF at a final concentration of 100 ng/ml; thelane at the left end of each electrophoretogram is the marker; thevertical axis of the graph plots the percentage of the amount of mRNAexpression for each exposure time of NGF, with the amount of mRNAexpression for exposure time zero being taken as 100%; the horizontalaxis plots the exposure time of NGF.

FIG. 26A shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of BDNF and the amount of ALPase mRNAexpression in HMS cells; it also shows an electrophoretogram depictingthe relationship with the amount of GAPDH mRNA expression; HMS cellswere all treated with BDNF at a final concentration of 100 ng/ml; thelane at the left end of each electrophoretogram is the marker; thevertical axis of the graph plots the percentage of the amount of mRNAexpression for each exposure time of BDNF, with the amount of mRNAexpression for exposure time zero being taken as 100%; the horizontalaxis plots the exposure time of BDNF.

FIG. 26B shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of BDNF and the amount of OCN mRNA expressionin HMS cells; it also shows an electrophoretogram depicting therelationship with the amount of GAPDH mRNA expression; HMS cells wereall treated with BDNF at a final concentration of 100 ng/ml; the lane atthe left end of each electrophoretogram is the marker; the vertical axisof the graph plots the percentage of the amount of mRNA expression foreach exposure time of BDNF, with the amount of mRNA expression forexposure time zero being taken as 100%; the horizontal axis plots theexposure time of BDNF.

FIG. 26C shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of BDNF and the amount of OPN mRNA expressionin HMS cells; it also shows an electrophoretogram depicting therelationship with the amount of GAPDH mRNA expression; HMS cells wereall treated with BDNF at a final concentration of 100 ng/ml; the lane atthe left end of each electrophoretogram is the marker; the vertical axisof the graph plots the percentage of the amount of mRNA expression foreach exposure time of BDNF, with the amount of mRNA expression forexposure time zero being taken as 100%; the horizontal axis plots theexposure time of BDNF.

FIG. 26D shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of BDNF and the amount of BSP mRNA expressionin HMS cells; it also shows an electrophoretogram depicting therelationship with the amount of GAPDH mRNA expression; HMS cells wereall treated with BDNF at a final concentration of 100 ng/ml; the lane atthe left end of each electrophoretogram is the marker; the vertical axisof the graph plots the percentage of the amount of mRNA expression foreach exposure time of BDNF, with the amount of mRNA expression forexposure time zero being taken as 100%; the horizontal axis plots theexposure time of BDNF.

FIG. 26E shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of BDNF and the amount of type I collagen mRNAexpression in HMS cells; it also shows an electrophoretogram depictingthe relationship with the amount of GAPDH mRNA expression; HMS cellswere all treated with BDNF at a final concentration of 100 ng/ml; thelane at the left end of each electrophoretogram is the marker; thevertical axis of the graph plots the percentage of the amount of mRNAexpression for each exposure time of BDNF, with the amount of mRNAexpression for exposure time zero being taken as 100%; the horizontalaxis plots the exposure time of BDNF.

FIG. 27A shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of NT-3 and the amount of ALPase mRNAexpression in HMS cells; it also shows an electrophoretogram depictingthe relationship with the amount of GAPDH mRNA expression; HMS cellswere all treated with NT-3 at a final concentration of 100 ng/ml; thelane at the left end of each electrophoretogram is the marker; thevertical axis of the graph plots the percentage of the amount of mRNAexpression for each exposure time of NT-3, with the amount of mRNAexpression for exposure time zero being taken as 100%; the horizontalaxis plots the exposure time of NT-3.

FIG. 27B shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of NT-3 and the amount of OCN mRNA expressionin HMS cells; it also shows an electrophoretogram depicting therelationship with the amount of GAPDH mRNA expression; HMS cells wereall treated with NT-3 at a final concentration of 100 ng/ml; the lane atthe left end of each electrophoretogram is the marker; the vertical axisof the graph plots the percentage of the amount of mRNA expression foreach exposure time of NT-3, with the amount of mRNA expression forexposure time zero being taken as 100%; the horizontal axis plots theexposure time of NT-3.

FIG. 27C shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of NT-3 and the amount of OPN mRNA expressionin HMS cells; it also shows an electrophoretogram depicting therelationship with the amount of GAPDH mRNA expression; HMS cells wereall treated with NT-3 at a final concentration of 100 ng/ml; the lane atthe left end of each electrophoretogram is the marker; the vertical axisof the graph plots the percentage of the amount of mRNA expression foreach exposure time of NT-3, with the amount of mRNA expression forexposure time zero being taken as 100%; the horizontal axis plots theexposure time of NT-3.

FIG. 27D shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of NT-3 and the amount of BSP mRNA expressionin HMS cells; it also shows an electrophoretogram depicting therelationship with the amount of GAPDH mRNA expression; HMS cells wereall treated with NT-3 at a final concentration of 100 ng/ml; the lane atthe left end of each electrophoretogram is the marker; the vertical axisof the graph plots the percentage of the amount of mRNA expression foreach exposure time of NT-3, with the amount of mRNA expression forexposure time zero being taken as 100%; the horizontal axis plots theexposure time of NT-3.

FIG. 27E shows by an electrophoretogram and a bar graph the relationshipbetween the exposure time of NT-3 and the amount of type I collagen mRNAexpression in HMS cells; it also shows an electrophoretogram depictingthe relationship with the amount of GAPDH mRNA expression; HMS cellswere all treated with NT-3 at a final concentration of 100 ng/ml; thelane at the left end of each electrophoretogram is the marker; thevertical axis of the graph plots the percentage of the amount of mRNAexpression for each exposure time of NT-3, with the amount of mRNAexpression for exposure time zero being taken as 100%; the horizontalaxis plots the exposure time of NT-3.

FIG. 28 is a bar graph showing the effects of ascorbic acid (Aa), NGF,BDNF and NT-3 on the proliferation of HMS cells; the vertical axis ofthe graph plots the percentage of the absorbance of each test group asrelative to the control group; the vertical lines on the bars in thegraph represent the range of mean±standard deviation; * and ** meanp<0.05 and p<0.01, respectively (statistical testing by t-test).

FIG. 29A is an optical microscopic view (×20) of a bone defect atfurcation which was packed with a transplant containing NGF (100 μg/ml),prepared in Example 8.

FIG. 29B is a microscopic view (×20) of a bone defect at furcation whichwas packed with a transplant containing NT-3 (100 μg/ml), prepared inExample 8.

BEST MODES FOR CARRYING OUT THE INVENTION

On the following pages, more specific embodiments of the presentinvention and methods for carrying out the invention are described.

The term “periodontal tissue” as used herein means a tissue composed ofthe gingiva, alveolar bone, periodontal ligament (periodontal membrane),and cementum.

“Gingiva” means the soft tissue that covers the cervical root surfaceand part of the alveolar bone; it consists of the gingival epitheliumand the gingival lamina propria.

“Periodontal ligament” means the connective tissue that bridges thealveolar bone and the cementum and is also known as the periodontalmembrane.

“Alveolar bone” is classified into the alveolar bone propercorresponding to the compact bone of the alveolar wall surrounding thedental root and the outer supporting alveolar bone consisting of thespongiosa and the compact bone.

“Cementum” is the hard tissue in the outermost layer of the dental rootand is classified into the cellular cementum that contains cementocytesand the acellular cementum that has no cementocytes.

“Dental pulp” is a tissue that controls the vital reactions of the teethand forms dentin as it reacts to physiological or pathological stimuli.It is composed of pulp cells, nerve fibers, extracellular matrix, bloodvessels, etc.

“Regeneration” means reconstruction and reproduction of lost tissues,destroyed tissues and damaged tissues; “regeneration of the periodontaltissue” means restoring the periodontal tissue to an initial state sothat it functions properly.

“Repair” means healing of a wounded tissue as in case where in, thestructure and functions of the wound have not yet been fully restored;and “repair of the periodontal tissue” includes the formation of anepithelial attachment to the dental root surface.

“Preventing apical invasion of gingival epithelium along the dental rootsurface” means preventing gingival epithelium cells from growing towardthe root apex along the dental root surface.

“Transplant for periodontal tissue regeneration” is a material thatenhances the regeneration of the periodontal tissue. In order to causeneurotrophic factors such as BDNF to act on a given in vivo site (e.g. amissing site of the alveolar bone) at a specified concentration, acertain scaffold is necessary. A material working as such a scaffold iscombined with a neurotrophic factor such as BDNF to provide thetransplant of the present invention.

“Periodontal diseases” means inflammatory diseases involving theperiodontal tissue that are caused by localized bacteria and the like.

“Repaired dentin” means dentin formed as the result of external stimuli.

“Pulpal diseases” means inflammatory diseases, retroplasia, etc. of thedental pulp.

The present invention is applicable to warm-blooded animals such ashumans and it is particularly preferred to apply to humans.

The neurotrophic factors such as BDNF, NGF, NT-3 and NT-4/5, which areto be used in the present invention, may be artificially produced bygene recombination or chemical synthesis; alternatively, they may be ofnative types.

The therapeutic agent for periodontal diseases of the present inventionis preferably applied topically in the form of a drug for externalapplication. If desired, the therapeutic agent may be filled into asyringe or the like and injected into a periodontal pocket. It is alsopossible to administer the therapeutic agent to a missing part of theperiodontal tissue during periodontal surgery. In this case, in order toassure a prolonged action at a specified concentration, it is alsopreferred to use the therapeutic agent of the present invention with anabsorbable material such as a sheet, or a sponge, etc. The therapeuticagent is preferably administered after removing the infected periodontaltissue. The therapeutic agent of the present invention can also beadministered locally in the form of an injection. For example, it may beinjected into the gingiva of a periodontal pocket or it may be injectedinto a cavity in the periodontal membrane near the alveolar crest. Itmay also be injected near the root apex.

The repaired dentin morphogenesis enhancer of the present invention ispreferably administered topically in the form of a drug for externalapplication. For example, the repaired dentin morphogenesis enhancer inliquid, cream, paste or other form may be applied to the pulp exposure;alternatively, it may be applied to the pulp amputation or extirpationof the pulp. If desired, a sheet or sponge impregnated with the activeingredient may be applied to provide temporary seal for a certainperiod. Alternatively, in the case of replanting a tooth that droppeddue to trauma or other cause, the enhancer may be applied to the rootapex or the like.

The therapeutic agent for periodontal diseases and the repaired dentinmorphogenesis enhancer according to the present invention may assume avariety of dosage forms including those for external application such ascream, ointment and lotion which are produced by conventionalpharmaceutical formulating procedures using pharmaceutically acceptablecarriers or diluents; they may also include injections based on aqueoussolvents. The therapeutic agent and the enhancer may also be in a powderdosage form and dissolved in a solubilizing fluid such as purified waterjust prior to use.

The therapeutic agent for periodontal diseases and the repaired dentinmorphogenesis enhancer according to the present invention may beadministered in doses that vary with the age of the subject, their sex,severity of the disease and other factors; in topical administration,normally, the dose is preferably in the range of 1×10⁻¹² g to 1×10⁻³ g,more preferably 1×10⁻¹¹ g to 1×10⁻⁷ g, most preferably 1×10⁻¹⁰ g to1×10⁻⁸ g, as a neurotrophic factor per tooth. Generally speaking, dosesfor local application by injection may be smaller than those used forexternal application.

The transplant of the present invention preferably contains 1×10⁻¹² g to1×10⁻³ g, more preferably 1×10⁻¹¹ g to 1×10⁻⁸ g, most preferably 1×10⁻¹⁰g to 1×10⁻⁹ g, of neurotrophic factors in a dose to be applied to onedefect at furcation.

The therapeutic agent for a periodontal disease, the repaired dentinmorphogenesis enhancer and the transplant according to the presentinvention may be used in combination with other drugs as long as theirefficacy is not impaired. BDNF, NGF, NT-3 and NT-4/5 may be used incombination with each other. They may also be used in combination withbone marrow derived mesemchymal stem cells (MSC), periodontal ligamentcells, gingival fibroblasts, vascular endothelial cells, etc. They mayalso be combined with calcium hydroxide preparations, antibacterialagents, etc.

The material to be combined with the neurotrophic factor in thetransplant of the present invention may be any material that causes nodamage to the living body and can maintain the neurotrophic factor atthe site to which it has been administered; preferred examples are aporous sheet and sponge. More preferred are biodegradable proteinmaterials (collagen, gelatin, albumin, and platelet-rich plasma (PRP))and tissue absorbing materials (polyglycolic acid (PGA), polylactic acid(PLA), poly(lactic acid-co-glycolic acid) (PLGA), hyaluronic acid (HA),and tricalcium phosphate (TCP)) since they need not be extracted later.Examples include TERUPLUG® (trade name of TERUMO CORPORATION), GCMembrane (trade name of GC Co., Ltd.) and Osferion (trade name ofOLYMPUS CORPORATION).

EXAMPLES

The present invention is described in greater detail by means of thefollowing examples.

Example 1

The effects of BDNF on human periodontal ligament cells (HPL cells) andhuman gingival keratinocytes (HGK) were investigated.

(1) Cells Used

(i) Human Periodontal Ligament Cells (HPL Cells)

HPL cells were separated from the periodontal ligament of a healthyhuman premolar that had been extracted for the sake of convenience inorthodontic treatment. In order to prevent the entrance of cells fromother connective tissues around the periodontal ligament, the healthyperiodontal ligament at the middle of the dental root excluding thecervical root surface and root apex of the extracted human premolar wasdetached with a scalpel and shredded. The shredded tissue was attachedto a cell culture Petri dish of 60 mm diameter (CORNING, N.Y.) andcultivated at 37° C. in a 5% CO₂ gas phase. The culture medium wasDulbecco's modified Eagle's medium (DMEM, NISSUI PHARMACEUTICAL CO.,LTD., Tokyo) supplemented with 10% FBS (GIBCO, Buffalo, N.Y.),penicillin (100 U/ml; MEIJI SEIKA KAISHA, LTD., Tokyo), streptomycin(100 μg/ml; MEIJI SEIKA KAISHA, LTD., Tokyo), and amphotericin B (1μg/ml; GIBCO); this culture medium is hereinafter designated “medium A”.The HPL cells in culture at passage 4-8 were used for the followingexperiment.

(ii) Human Gingival Keratinocytes (HGK)

Gingiva was obtained from patients after having their informed consentabout the need to perform experiments using human cultured cells and thepurpose of using the gingiva. The patients were suffering frompericoronitis of the wisdom teeth and when the causative wisdom teethwere being extracted, gingival pieces were acquired from an excessgingival flap. The obtained gingival pieces were treated with Dulbecco'sPBS(−) (PBS(−) of NISSUI PHARMACEUTICAL CO., LTD.) supplemented with0.01% ethylenediaminetetraacetic acid (EDTA) and 0.025% trypsin at 4° C.for 24 hours to separate HGK. Primary culture was performed on MCDB 153medium (Sigma) supplemented with bovine insulin (10 μg/ml; Sigma, St.Louis, Mo., USA), human transferrin (5 μg/ml; Sigma), 2-mercaptoethanol(10 μM), 2-aminoethanol (10 μM), sodium selenite (10 μM), bovinepituitary gland extract (50 μg/ml), penicillin (100 U/ml), streptomycin(100 μg/ml), and amphotericin B (50 ng/ml); this culture medium ishereinafter designated “medium C”. The culture was performed on a Petridish of 60 mm diameter (SUMILON CELTITE C-1 of SUMITOMO BAKELIGHTCOMPANY LIMITED, Tokyo) coated with bovine type I collagen at 37° C. ina 5% CO₂ gas phase. The HGK in culture at passage 3-4 were used for thefollowing experiment.

(2) Expression of BDNF and its Receptor in HPL Cells

The expression of mRNA for BDNF and TrkB in HPL cells and humanperiodontal ligament was investigated by reverse transcriptase PCR usinga 1st Strand cDNA Synthesis Kit for RT-PCR (Roche, Indianapolis).

The HPL cells obtained in (1)(i) above were recovered at the time whenthey reached confluence; the cells were then dissolved in ISOGEN (NipponGene, Tokyo) and centrifuged after adding chloroform; to the resultingaqueous phase, isopropanol was added to extract total RNA.

The human periodontal ligament obtained in (1)(i) above was homogenizedin ISOGEN and centrifuged after adding chloroform; to the resultingaqueous phase, isopropanol was added to extract total RNA.

Portions (1 μg each) of the purified total RNA were reverse transcribedusing oligo dT primers; the resulting cDNA was amplified by 30 cycles ofPCR and electrophoresed on 1.5% agarose gel. Glyceraldehyde 3-phosphatedehydrogenase (GAPDH) was used as a control.

The results are shown in FIG. 1. The control was glyceraldehyde3-phosphate dehydrogenase (GAPDH). As is clear from theelectrophoretograms, mRNAs of BDNF and TrkB were expressed in the humanperiodontal ligament and it was confirmed that mRNAs of BDNF and TrkBhad also been expressed in the HPL cells cultured after being separatedfrom the human periodontal ligament.

(3) Treatment of the Cells with BDNF

(i) HPL cells

The HPL cells obtained in (1)(i) above were cultivated on Petri dishesof 60 mm diameter (SUMILON CELTITE C-1) coated with bovine type Icollagen at 37° C. in a 5% CO₂ gas phase for 13 days at a density of3.5×10⁵ cells per Petri dish using medium A supplemented with 50 μg/mlof L-ascorbic acid. The culture medium used in this cultivation isdesignated “medium B”. The medium was changed once every two days. At 0,3, 6, 12 and 24 hours before the end of cultivation at day 14, the cellswere washed twice with DMEM and medium was changed for a serum-freeculture medium containing BDNF (Recombinant Human BDNF, R&D System,Minneapolis, USA) at a final concentration of 0, 1, 10, 50 or 100 ng/ml(the medium being DMEM supplemented with penicillin (100 U/ml),streptomycin (100 μg/ml), amphotericin B (1 μg/ml; GIBCO) and L-ascorbicacid (50 μg/ml)). This culture medium is designated “medium D”.

(ii) HGK

The HGK obtained in (1)(ii) above were inoculated on a 96-well plate(SUMILON CELTITE C-1 Plate 96F of SUMITOMO BAKELIGHT COMPANY LIMITED)coated with bovine type I collagen at a density of 2×10³ cells/well andcultivated at 37° C. in a 5% CO₂ gas using medium C. The medium waschanged once every two days. At day 4 or 5 in a cell proliferationphase, the cells on the plate were washed twice with MCDB 153 medium andmedium was changed for a culture medium containing BDNF at a finalconcentration of 0, 1, 10, 25, 50 or 100 ng/ml (the medium having thesame composition as medium C except that it did not contain a bovinepituitary extract). The cell was cultured for 24 hours.

(4) Expression of Bone-Related Proteins in HPL Cells

(i) Expression of mRNA

HPL cells were treated with BDNF at a final concentration of 0, 1, 10,50 or 100 ng/ml by the same procedure as described in (3)(i) above andtotal RNA was extracted from the thus treated HPL cells with ISOGEN andpurified. The expression of mRNA for alkali phosphatase (ALPase), bonemorphogenetic protein-2 (BMP-2), bone morphogenetic protein-4 (BMP-4),osteopontin (OPN) and osteoprotegerin (OPG) was quantitatively analyzedby monitoring the process of generation of PCR products in real-timeusing ABI PRISM 7700 (Applied Biosystems, Tokyo) (real-time PCR method).GAHPD was used as a control.

The results of measuring the time course effect of BDNF on theexpression of mRNA for the respective bone-related proteins are shown inFIGS. 2A-2C, 3A and 3B, and the results of measuring the dose effect ofBDNF are shown in FIGS. 4A-4C. In each graph, * and ** mean p<0.05 andp<0.01, respectively (statistical testing by t-test).

As is clear from those Figures, BDNF had no effect on the expression ofmRNA for OPG and BMP-4 but increased the amount of mRNA expression forALPase, BMP-2 and OPN in both a dose- and a time-dependent manner.

(ii) Expression of Proteins

The HPL cells obtained in (1)(i) above were seeded on a 48-well plate(SUMILON CELTITE C-1 Plate 48F of SUMITOMO BAKELITE COMPANY LIMITED)coated with bovine type I collagen at a density of 1×10⁴ cells/well andcultivated for 13 days using medium B. The medium was changed once everytwo days. Twenty-four hours before the end of cultivation at day 14, thecells on the plate were washed twice with DMEM and medium was changedfor serum-free medium D containing BDNF at a final concentration of 0,1, 10, 25, 50 or 100 ng/ml. After the end of culture, the supernatantwas recovered and the amounts of OPN and BMP-2 secreted in thesupernatant were measured by ELISA. For measuring the secreted OPN, asandwich ELISA kit (IBL, Gunma) was used, and a sandwich ELISA kit (R&DSystem) was used to measure the secreted BMP-2.

FIG. 5 shows the results of measuring the time course effect and thedose effect of BDNF on the secretion of OPN and BMP-2 in HPL cells. Ineach graph, * and ** mean p<0.05 and p<0.01, respectively (statisticaltesting by t-test). As is clear from FIG. 5, BDNF enhanced the secretionof OPN and BMP-2 in HPL cells.

(5) Proliferation of HPL Cells and HGK

The effects of BDNF on the ability of HPL cells and HGK to synthesizeDNA were measured by ELISA using a Cell Proliferation ELISA System,Version 2 (Amersham Pharmacia Biotech).

The HPL cells obtained in (1)(i) above were seeded on a 96-well plate(SUMILON CELTITE C-1 Plate 96F) coated with bovine type I collagen at adensity of 5×10³ cells/well and cultivated for 10 days using medium B.The cells were washed twice with DMEM and cultivated for 24 hours onmedium B (supplemented with 0.3% FBS instead of 10% FBS); thereafter,the medium was changed for a medium prepared by adding BDNF to the samemedium at a final concentration of 0, 1, 10, 25, 50 or 100 ng/ml; thecell was cultured for an additional 24 hours. Two hours before the endof the culture (viz. 22 hours after the addition of BDNF),bromodeoxyuridine (BrdU) was added to each well at a concentration of 10ng/ml so that it was incorporated into the cells. The culture wasperformed at 37° C. in a 5% CO₂ gas phase.

The HGK obtained in (1)(ii) above were cultivated and treated with BDNFby the same procedures as in (3)(ii) above. Two hours before the end ofthe culture (viz. 22 hours after the addition of BDNF),bromodeoxyuridine (BrdU) was added to each well at a concentration of 10ng/ml so that it was incorporated into the cells.

After the end of culture, the HPL cells and HGK were fixed and thenblocking was performed; a peroxidase labeled anti-BrdU antibody wasallowed to act on the cells at room temperature for 2 hours and a TMB(3,3′,5,5′-tetramethylbenzidine) substrate was added to measure theabsorbance at a wavelength of 450 nm with an absorptiometer (MICRO PLATEREADER, TOSOH). As a control, cells on which basic fibroblast growthfactor (bFGF) had been allowed to act at final concentrations of 0, 0.3,1, 3, 5 and 10 ng/ml for 24 hours were treated in the same way so as tomeasure their ability to synthesize DNA.

The results are shown in FIG. 6; (A) is a bar graph showing the effectson HPL cells, and (B) is a bar graph showing the effects on HGK. In eachgraph, * and ** mean p<0.05 and p<0.01, respectively (statisticaltesting by t-test).

As is clear from FIG. 6, BDNF enhanced the DNA synthesizing ability ofHPL cells but had no effect on the DNA synthesizing ability of HGK.

(6) Collagen Synthesis by HPL Cells

The HPL cells obtained in (1)(i) above were seeded on a 48-well platecoated with bovine type I collagen and cultivated for 13 days usingmedium B. The medium was changed once every two days. At 0, 3, 6, 12 or24 hours before the end of cultivation at day 14, the cells on the platewere washed twice with DMEM and medium was changed for serum-free mediumD containing BDNF at a final concentration of 0, 1, 10, 25, 50 or 100ng/ml.

Using a Procollagen type I C-peptide (PIP) EIA Kit (TAKARA), the amountin which the HPL cells synthesized collagen was measured by ELISA. Usinga monoclonal antibody (peroxidase-labeled) specific to type Iprocollagen C-terminal propeptide (PIP), the amount of collagensynthesized in the supernatant of the culture of HPL cells wasdetermined by measuring the absorbance at a wavelength of 450 nm with anabsorptiometer (MICRO PLATE READER).

The results are shown in FIG. 7; (A) shows the results of measuring thedose effect of BDNF on the synthesis of type I collagen, and (B) showsthe results of measuring the time course effect. As is clear from FIG.7, BDNF increased the amount of type I collagen synthesized by HPLcells.

Example 2

The effect of BDNF on beagle dogs as a model of class III furcationdefect was investigated.

TERUPLUG® (trade name of TERUMO CORPORATION) of 8 mm diameter×5 mm wasimpregnated with 25 μl each of BDNF solutions (in sterile physiologicalsaline) at concentrations of 5, 25 and 50 μg/ml to prepare transplants.

Seven female beagle dogs (12-20 months old, weighing 10-14 kg) weresubjected to scaling and root planning of the teeth with a hand scalerwhile they were under sedation with intramuscularly injected DOMITOR(MEIJI SEIKA KAISHA, LTD.) Thereafter, at a frequency of once every twodays, the oral cavity of each animal was brushed and cleaned by thegargle ISOJIN (trade name of MEIJI SEIKA KAISHA, LTD.) containingpovidone iodine as an active ingredient; this practice was continued fora month to establish a clinically healthy periodontal tissue in eachanimal.

The beagle dogs were subjected to general anesthesia by intravenousinjection of a pentobarbital-containing anesthetic and local infiltratedanesthesia was applied to the mandibular buccal gingiva on both theright and left sides; the gingival sulcus was incised between the distalpart of the first premolar and the mesial part of the first molar andthe gingiva was detached to form a mucoperiosteal flap. Subsequently,the alveolar bones at the furcation regions of the second, third andfourth premolars on both the right and left sides were removed with around bur and a bone chisel to prepare bone defects at furcation ofclass III (according to the classification by Lindhe & Nyman). The sizeof each bone defect was such that it extended from the area beneath theuntreated furcation to about 4 mm toward the root apex.

The residual cementum on the exposed dental root surface was removedwith a hand scaler and the interior of each bone defect at the furcationwas thoroughly washed with physiological saline to rinse off the debris,followed by packing with the TERUPLUG® transplant of 8 mm diameter×5 mmper site. A TERUPLUG® transplant of 8 mm diameter×5 mm that did notcontain BDNF but which was only impregnated with 25 μl of sterilephysiological saline was packed into the same bone defect to prepare acontrol.

Six weeks after the operation, the animals were systemically perfusedwith 4% paraformaldehyde under general anesthesia by intravenousinjection of a pentobarbital-containing anesthetic. After the perfusion,the mandible of each animal was dissected and the treated teeth andperiodontal tissue were extracted en bloc. The obtained sample was fixedwith 4% paraformaldehyde, decalcified with 10% EDTA, followed bydehydration with graded alcohol and embedding in paraffin in accordancewith the usual practice. From this specimen, serial sections (about 5 μmthick) were cut in the mesial-distal plane through the buccal-lingualextension of the tooth and they were stained with hematoxylin and eosin(H&E).

Among the thus prepared tissue specimens, those which were cut in themesial-distal plane through the buccal-lingual extension of the toothand which had been cut near the midroot were chosen and subjected totissue examination and measurement with an optical microscope (ECLIPSEE600, NIKON). The percent bone regeneration was expressed as the ratio(in percentage) of the area of the regenerated alveolar bone to the areaof the exposed defect at furcation. The percent cementum regenerationwas expressed as the ratio (in percentage) of the length of theregenerated cementum to the length of the exposed dental root surface.

The results are shown in FIGS. 8, 9A, 9B and 10. FIG. 8 (A) shows theresults of measuring the effect of BDNF on the regeneration of cementum;FIG. 8 (B) shows the results of measuring the effect of BDNF on theregeneration of alveolar bone. FIG. 9A shows a hematoxylin-eosin stainedspecimen of the bone defect at furcation which was not administered BDNF(control), and FIG. 9B is an optical microscopic view (×20) of the bonedefect at furcation which was administered BDNF (packed with thetransplant containing BDNF (5 μg/ml)). FIG. 10 is a partial enlargedview (×200) of the area just beneath the furcation in FIG. 9B.

As is clear from FIG. 8, when administered BDNF, the dog model of classIII furcation defect had discernible regeneration of the cementum andthe alveolar bone.

In the control specimen shown in FIG. 9A, some degree of regenerationwas observed in the cementum, alveolar bone and periodontal ligament butit merely extended from the bottom of the bone defect to approximatelyone half the way toward the corona. In the defective area right underthe furcation, there was no discernible regeneration of the cementum andthe alveolar bone but there was observed an invasion of the epithelium;that area was practically occupied with a connective tissue mainlyconsisting of fibroblasts, collagen fibers and blood vessels.

In the specimen of the bone defect at furcation which was administeredBDNF shown in FIGS. 9B and 10, the cementum was regenerated in almostall parts of the exposed dental root surface and there was nodiscernible invasion of the epithelium. In addition, a periodontalligament maintaining a certain width was observed between theregenerated cementum and the regenerated alveolar bone.

Example 3

The effects of NGF on HPL cells and HGK were investigated.

(1) Expression of NGF and its Receptor in HPL Cells

By the same procedures as in Example 1(2) described above, total RNA wasrecovered from HPL cells and purified. With the obtained total RNA beingused as a sample, the expression of mRNA for NGF and TrkA was measuredby Northern blotting. GAPDH was used as a control.

The results are shown in FIGS. 11A and 11B.

FIG. 11A shows the expression of mRNA for NGF, and FIG. 11B shows theexpression of mRNA for TrkA. As is clear from the Figures, it wasconfirmed that mRNA of NGF and mRNA of TrkA had been expressed in theHPL cells.

(2) Expression of Bone-Related Proteins in HPL Cells

The effects of NGF on the expression of mRNA for bone-related proteinsin HPL cells were investigated.

HPL cells were treated by the same procedure as in Example 1(4)(i)except that BDNF was replaced by NGF (Recombinant Human NGF, R&D System,Minneapolis, USA) at final concentrations of 0, 5, 10, 25, 50 and 100ng/ml. The amounts in which the NGF-treated HPL cells expressed themRNAs of ALPase, BMP-2 and OPN were measured by the same method as inExample 1(4)(i).

FIGS. 12, 13 and 14 show the results of measuring the time course effectand the dose effect of NGF on the expression of mRNA for OPN, ALPase andBMP-2, respectively. In each graph, * and ** mean p<0.05 and p<0.01,respectively. Testing was done by t-test.

As is clear from FIGS. 12, 13 and 14, NGF increased the expression ofmRNA for ALPase, BMP-2 and OPN in both a dose- and a time-dependentmanner.

(3) Proliferation of HPL Cells and HGK

The effects of NGF on the ability of HPL cells and HGK to synthesize DNAwere measured.

HPL cells and HGK were treated by the same method as in Example 1(5)except that BDNF was replaced by NGF at final concentrations of 0, 5,10, 25, 50 and 100 ng/ml. The ability of the NGF-treated HPL cells andHGK to synthesize DNA was measured by the same method as in Example1(5).

The results are shown in FIG. 15. The exposure time of NGF was 24 hoursin all runs; (A) shows the effects on HPL cells, and (B) shows theeffects on HGK. In each graph, * and ** mean p<0.05 and p<0.01,respectively. Testing was done by t-test.

As is clear from FIG. 15, NGF enhanced the DNA synthesizing ability ofHPL cells but lowered the DNA synthesizing ability of HGK.

Example 4

The effects of NT-3 on HPL cells and HGK were investigated.

(1) Expression of NT-3 and its Receptor in HPL Cells

By the same procedures as in Example 1(2) described above, total RNA wasrecovered from HPL cells and purified. With the obtained total RNA beingused as a sample, the expression of mRNA for NT-3 and TrkC was measuredby Northern blotting. GAPDH was used as a control.

The results are shown in FIGS. 16A and 16B. FIG. 16A shows theexpression of mRNA for NT-3, and FIG. 16B shows the expression of mRNAfor TrkC. As is clear from the Figures, it was confirmed that mRNA ofNT-3 and mRNA of TrkC had been expressed in the HPL cells.

(2) Expression of Bone-Related Protein in HPL Cells

The effects of NT-3 on the ALPase activity in HPL cells wereinvestigated.

HPL cells were treated by the same procedure as in Example 1(3)(i)except that BDNF was replaced by NT-3 (Recombinant Human NT-3, R&DSystem, Minneapolis, USA) at final concentrations of 0, 1, 10 and 50ng/ml, and their ALPase activity was quantitated in accordance with theBessey-Lowry method. Stated more specifically, the NT-3 treated HPLcells were washed three times with a phosphate buffer and after adding10 mM Tris-HCl buffer, the cells were sonicated under ice cooling toprepare a sample. The ALPase activity in the sample was measured with anALPase measuring kit (Wako Pure Chemical Industries, Ltd.) usingp-nitrophenyl phosphate as a substrate.

FIG. 17 shows the results of measuring the dose effect of NT-3 on ALPaseactivity. The exposure time of NT-3 was 24 hours in all runs. As isclear from the Figure, NT-3 did not have much effect on ALPase activity.

(3) Proliferation of HPL Cells

HPL cells separated by the same method as in Example 1(1) were treatedby the same method as in Example 1(5) except that BDNF was replaced byNT-3 at final concentrations of 0, 1, 5, 10, 50 and 100 ng/ml. Theirability to synthesize DNA was measured by the same method as in Example1(5).

The results are shown in FIG. 18. The exposure time of NT-3 was 24 hoursin all runs. In the graph, * and ** mean p<0.05 and p<0.01,respectively. Testing was done by t-test. As is clear from the Figure,NT-3 enhanced the DNA synthesizing ability of HPL cells.

Example 5

The expression of mRNA for bone-related proteins by T-4/5 in humanperiodontal ligament cells (HPL cells) was investigated.

(1) Treatment of HPL Cells with NT-4/5

The HPL cells obtained in Example 1(1)(i) above were cultivated on Petridishes of 60 mm diameter (SUMILON CELTITE C-1) coated with bovine type Icollagen at 37° C. in a 5% CO₂ gas phase for 13 days at a density of3.5×10⁵ cells per Petri dish using medium B (50 μg/ml). The medium waschanged once every two days. At 0, 3, 6, 12 and 24 hours before the endof cultivation at day 14, the cells were washed twice with DMEM andmedium was changed for medium D containing NT-4/5 (R&D) at a finalconcentration of 50 ng/ml.

(2) Expression of mRNA in HPL Cells

HPL cells were treated with NT-4/5 at a final concentration of 50 ng/mlby the same procedure as described in (3)(i) above and total RNA wasextracted from the thus treated HPL cells with ISOGEN and purified. Theexpression of mRNA for ALPase, BMP-2, OPN, osteocalcin (OCN), BMP-7,BMP-4 and OPG was quantitatively analyzed by monitoring the process ofgeneration of PCR products in real-time using ABI PRISM 7700 (AppliedBiosystems, Tokyo) (real-time PCR method). GAPDH was used as a control.

The results of measuring the time course effect of NT-4/5 on theexpression of mRNA for the respective bone-related proteins are shown inFIGS. 19A, 19B and 19C. In each graph, * and ** mean p<0.05 and p<0.01,respectively (statistical testing by t-test). As is clear from thoseFigures, NT-4/5 enhanced the expression of mRNA for OPN, BMP-2, ALPase,OCN and BMP-7 in HPL cells but had no effect on the expression of mRNAfor BMP-4 and OPG (data not shown).

Example 6

The effects of NGF, BDNF, NT-3 and NT-4/5 on human pulp cells (HP cells)were investigated.

(1) Cells Used

Healthy dental pulp that had been obtained for the sake of conveniencein dental pulp removal was shredded. The shredded tissue was attached toa cell culture Petri dish of 60 mm diameter (CORNING, N.Y.) andcultivated on medium A at 37° C. in a 5% CO₂ gas phase. HP cells inculture at passage 4-8 were used for the following experiment.

(2) Treatment of the Cells with NGF, BDNF, NT-3 and NT-4/5

NGF, BDNF, NT-3 or NT-4/5 was added to medium D at final concentrationsof 0, 5, 10, 25, 50 and 100 ng/ml to prepare culture mediums containingthose neurotrophic factors at the indicated concentrations. The HP cellsobtained in (1) above were cultivated on Petri dishes of 60 mm diameter(SUMILON CELTITE C-1) coated with bovine type I collagen at 37° C. in a5% CO₂ gas phase for 13 days at a density of 3.5×10⁵ cells per Petridish, using medium B. The medium was changed once every two days.Twenty-four hours before the end of cultivation at day 14, the cellswere washed twice with DMEM and medium was changed for either one of theneurotrophic factors containing culture mediums.

(3) Expression of mRNA in HP Cells

From the HP cells of (1) above treated with NGF, BDNF, NT-3 or NT-4/5 atthe indicated concentrations for 24 hours, total RNA was extracted usingISOGEN and purified.

The expression of mRNA for ALPase, BMP-2, dentin sialophosphoprotein(DSPP), type I collagen, OPN and OCN was quantitatively analyzed bymonitoring the process of generation of PCR products in real-time usingABI PRISM 7700 (Applied Biosystems, Tokyo) (real-time PCR method). GAPDHwas used as a control.

The results of measuring the dose effects of NGF, BDNF, NT-3 and NT-4/5on the expression of mRNA for the respective bone-related proteins areshown in FIGS. 20, 21, 22 and 23, respectively. As is clear from thoseFigures, NGF, BDNF, NT-3 and NT-4/5 enhanced the expression of mRNA forALPase, BMP-2, DSPP, OPN and OCN in HP cells. BDNF and NT-4/5 alsoenhanced the expression of mRNA for type I collagen.

(4) Proliferation of HP Cells

The effects of NGF, BDNF, NT-3 and NT-4/5 on the ability of HP cells tosynthesize DNA were measured by ELISA using a Cell Proliferation ELISASystem, Version 2 (Amersham Pharmacia Biotech).

To medium B supplemented with 0.3% FBS instead of 10% FBS, each of NGF,BDNF, NT-3 and NT-4/5 was added at final concentrations of 0, 5, 10, 25,50 and 100 ng/ml to prepare culture mediums containing thoseneurotrophic factors.

The HP cells obtained in (1) above were seeded on a 96-well plate(SUMILON CELTITE C-1 Plate 96F) coated with bovine type I collagen at adensity of 5×10³ cells/well and cultivated for 10 days using medium B.The cells were washed twice with DMEM and cultivated for 24 hours onmedium B, except that it was supplemented with 0.3% FBS instead of 10%FBS; thereafter, the medium was changed for either one of theabove-described neurotrophic factors containing culture mediums, andculture was continued for an additional 24 hours. Two hours before theend of the culture (viz. 22 hours after the addition of the neurotrophicfactors), bromodeoxyuridine (BrdU) was added to each well at aconcentration of 10 ng/ml so that it was incorporated into the cells.Culture was performed at 37° C. in a 5% CO₂ gas phase.

After the end of culture, the HP cells were fixed and then blocking wasperformed; a peroxidase labeled anti-BrdU antibody was allowed to act onthe cells at room temperature for 2 hours and a TMB(3,3′,5,5′-tetramethylbenzidine) substrate was added to measure theabsorbance at a wavelength of 450 nm with an absorptiometer (MICRO PLATEREADER, TOSOH).

The results are shown in FIG. 24, from which it is clear that NGF, BDNF,NT-3 and NT-4/5 enhanced the DNA synthesizing ability of HP cells.

Example 7

The effects of NGF, BDNF, NT-3 and ascorbic acid on human mesenchymalstem cells (HMS cells) were investigated.

(1) Cells Used

HMS cells were separated in accordance with the method of Tsutsumi etal. (S. Tsutsumi: BBRC, 26, 288(2), 2001). To be specific, when a wisdomtooth was removed from patients who gave a fully informed consent to theexperiment, the mandibular bone was punctured to aspirate bone marrow.The bone marrow was immediately mixed with Dulbecco's modified Eagle'smedium (DMEM, Sigma, USA) supplemented with heparin sodium (200 U/ml,Sigma, USA) and the mixture was centrifuged (150 g, 5 min). After thecentrifugation, the supernatant was removed and the resulting cellcomponent was suspended in DMEM containing 10% fetal calf serum (FCS,Biological Industries, Israel), 100 units/ml of penicillin (MEIJI SEIKAKAISHA, LTD., Tokyo), 100 μg/ml of streptomycin (MEIJI SEIKA KAISHA,LTD., Tokyo) and 1 μg/ml of amphotericin B (GIBCO, USA) and thesuspension was seeded on cell culture Petri dishes of 100 mm diameter(Corning, USA) such that the bone marrow was at 200-500 μl/dish and themedium was at 10 ml/dish. Culture was performed at 37° C. in a 5% CO₂gas phase. Subsequently, medium change was done every four days. Justbefore the proliferating cells had reached confluence, phosphatebuffered physiological saline (PBS, NISSUI PHARMACEUTICAL CO., LTD.,Tokyo) containing 0.05% trypsin (Difco, USA), 0.02% EDTA (KATAYAMACHEMICAL INDUSTRIES Co., Ltd., Osaka), 100 units/ml of penicillin and100 μg/ml of streptomycin was used to disperse the cells. The cells thusdispersed were suspended in DMEM supplemented with 20% FCS, 10% dimethylsulfoxide (DMSO, KATAYAMA CHEMICAL INDUSTRIES Co., Ltd., Osaka), 100units/ml of penicillin and 100 μg/ml of streptomycin; the suspended celldensity was 1.0×10⁶ cells/ml; the suspension was distributed in 1-mlportions among serum tubes (SUMITOMO BAKELITE COMPANY LIMITED, Tokyo),cooled at −20° C. for 2 hours, then at −80° C. overnight before storagein liquid nitrogen.

(2) Expression of mRNA for Bone-Related Proteins in HMS Cells

(i) Treatment of Cells with NGF, BDNF and NT-3

The HMS cells obtained in (1) above were suspended in 10% FCS containingDMEM (supplemented with 100 units/ml of penicillin (MEIJI SEIKA KAISHA,LTD., Tokyo), 100 μg/ml of streptomycin (MEIJI SEIKA KAISHA, LTD.,Tokyo) and 1 μg/ml of amphotericin B (GIBCO, USA)) and the suspensionwas seeded on a 6-well cell culture plate at a density of 1.0×10⁵cells/well. The cells were cultivated for a week and just before theyhad reached confluence, the medium was changed for FCS-free DMEM(supplemented with 100 units/ml of penicillin (MEIJI SEIKA KAISHA, LTD.,Tokyo), 100 μg/ml of streptomycin (MEIJI SEIKA KAISHA, LTD., Tokyo) and1 μg/ml of amphotericin B (GIBCO, USA)) and either one of NGF, BDNF andNT-3 was allowed to act for 12 or 24 hours at a concentration of 100ng/ml. After the end of culture, total RNA was extracted using ISOGEN(trade name).

(ii) Expression of mRNA

PCR was performed using primers specific to ALPase, OCN, OPN, bonesialoprotein (BSP) and type I collagen. The PCR consisted ofdenaturation at 94° C. for 2 minutes, 30 cycles of 94° C.×15 seconds,annealing for 30 seconds and 72° C.×50 seconds (but 35 cycles in thecase of BSP), followed by extension at 72° C. for 7 minutes. The PCRproducts obtained were electrophoresed on a 2% agarose gel containing0.002% ethidium bromide. The density of the bands after electrophoresiswas measured using NIH image.

The results are shown in FIGS. 25A-25E, 26A-26E and 27A-27E. As is clearfrom the Figures, NGF had no marked effect on the expression of mRNA forany of ALPase, OCN, OPN, BSP and type I collagen in HMS cells. BDNFhighly enhanced the expression of mRNA for ALPase, OPN, BSP and BMP-2while enhancing the expression of the OCN gene to some extent. NT-3enhanced the expression of mRNA for ALPase and type I collagen.

(3) The Effects of Ascorbic Acid, NGF, BDNF and NT-3 on theProliferation of HMS Cells

The HMS cells obtained in (1) above were suspended in 10% FCS containingDMEM (product of NISSUI PHARMACEUTICAL CO., LTD.; supplemented with 100units/ml of penicillin (MEIJI SEIKA KAISHA, LTD., Tokyo), 100 μg/ml ofstreptomycin (MEIJI SEIKA KAISHA, LTD., Tokyo) and 1 μg/ml ofamphotericin B (GIBCO, USA)) and the suspension was seeded on a 96-wellcell culture plate (Corning, USA) at a density of 5.0×10³ cells/well. Inthe test groups, 50 μg/ml of ascorbic acid (Sigma, USA) or 100 ng/ml ofNGF (FUNAKOSHI, Tokyo) or 100 ng/ml of BDNF (FUNAKOSHI, Tokyo) or 100ng/ml of NT-3 (FUNAKOSHI, Tokyo) was singly added to the culture medium24 hours after the start of culture, and cultivation was continued for 7more days. Medium change was done on day 4. The control group wascultivated on 10% FCS containing DMEM (product of NISSUI PHARMACEUTICALCO., LTD.; supplemented with 100 units/ml of penicillin (MEIJI SEIKAKAISHA, LTD., Tokyo), 100 μg/ml of streptomycin (MEIJI SEIKA KAISHA,LTD., Tokyo) and 1 μg/ml of amphotericin B (GIBCO, USA)). After 7 daysof culture, the mediums were entirely changed for 10% FCS containingDMEM (supplemented with 100 units/ml of penicillin (MEIJI SEIKA KAISHA,LTD., Tokyo), 100 μg/ml of streptomycin (MEIJI SEIKA KAISHA, LTD.,Tokyo) and 1 μg/ml of amphotericin B (GIBCO, USA)) and the number ofviable cells was counted by absorbance measurement at 490 nm withCellTiter 96 (trade name) AQueous One Solution Cell Proliferation AssayKit (Promega, USA).

The results are shown in FIG. 28. The vertical axis of the graph plotsthe percentage of the absorbance in the test groups as against thecontrol group. In the graph, * and ** mean p<0.05 and p<0.01,respectively (statistical testing by t-test). As is clear from theFigure, the HMS cells cultivated on the medium supplemented with eitherone of ascorbic acid, NGF, BDNF and NT-3 showed a significantly highertendency to proliferate than the control. In particular, theproliferation enhancing effect of ascorbic acid, BDNF and NT-3 wasstronger than that of NGF.

Example 8

The effects of NGF and NT-3 on beagle dogs as models of class IIIfurcation defect were investigated.

Experiments were conducted as in Example 2, except that TERUPLUG® of 8mm diameter×5 mm impregnated with 25 μl of a NGF solution (in sterilephysiological saline) at a concentration of 100 μg/ml instead of theBDNF solutions (in sterile physiological saline) at concentrations of 5,25 and 50 μg/ml, as well as TERUPLUG® of the same size which wasimpregnated with 25 μl of a NT-3 solution (in sterile physiologicalsaline) at a concentration of 100 μg/ml were used as transplants. Amongthe tissue specimens prepared (and hematoxylin-eosin stained), thosewhich were cut in the mesial-distal plane through the buccal-lingualextension of the tooth and which had been cut near the midroot werechosen and examined under an optical microscope (ECLIPSE E600, NIKON).

FIG. 29A is an optical microscopic view of the bone defect at furcationpacked with the NGF containing transplant, and FIG. 29B is an opticalmicroscopic view (×20) of the bone defect at furcation packed with theNT-3 containing transplant. As is clear from the microphotographs,regenerated bone was observed in the dog models of class III furcationdefect in response to the administration of NGF or NT-3.

INDUSTRIAL APPLICABILITY

The therapeutic agent for periodontal diseases, the repaired dentinmorphogenesis enhancer, the therapeutic method, the transplant forperiodontal tissue regeneration, and the method for regenerating theperiodontal tissue according to the present invention have a potentialto become effective in the treatment of periodontal diseases andendodontic therapy.

1.-29. (canceled)
 30. A repaired dentin morphogenesis enhancer whichcomprises a neurotrophic factor as an active ingredient, wherein theneurotrophic factor is a brain-derived neurotrophic factor orneurotrophin-4/5.
 31. A therapeutic agent for a pulpal disease whichcomprises a neurotrophic factor as an active ingredient, wherein theneurotrophic factor is a brain-derived neurotrophic factor orneurotrophin-4/5.
 32. A therapeutic method for pulpal disease whichcomprises administering a therapeutically effective amount of aneurotrophic factor to a subject who is suffering or prone to sufferfrom the disease in order to enhance morphogenesis of repaired dentin,wherein the neurotrophic factor is a brain-derived neurotrophic factoror neurotrophin-4/5.
 33. The method according to claim 32 whichcomprises administering to the subject a repaired dentin morphogenesisenhancer which comprises a neurotrophic factor as an active ingredient,wherein the neurotrophic factor is a brain-derived neurotrophic factoror neurotrophin-4/5.
 34. The method according to claim 32 whichcomprises administering to the subject a therapeutic agent for a pulpaldisease which comprises a neurotrophic factor as an active ingredient,wherein the neurotrophic factor is a brain-derived neurotrophic factoror neurotrophin-4/5.
 35. The method according to claim 32 wherein therepaired dentin is added to the inner surfaces of the pulp cavity.